Diagnostic agents

ABSTRACT

Diagnostic agents are described for determining high density lipoprotein cholesterol in body fuids such as serum or plasma. One is an enzymatic reagent for quantitative analysis for cholesterol composed of cholesterol oxidase, cholesterol esterase, peroxidase, 4-amino-antipyrine, a phenol or phenol derivative, a surfactant, such as sodium cholate and a polyglycol of molecular weight from about 190 to 1000, buffered to pH 5.5 to 7.8 in water. The other is a single, stable precipitating agent for lipoproteins in body fluids, comprising a bivalent metal salt of a monobasic acid, such as magnesium chloride and a water-soluble polyanion, such as sodium phosphotungstate in water at pH 5 to 8.

This application is a continuation of Ser. No. 108,846, Dec. 31, 1979,abandoned which is a division of Ser. No. 899,597, Apr. 24, 1978, nowU.S. Pat. No. 4,226,713.

This invention relates to a diagnostic reagent for total and highdensity lipoprotein (HDL) cholesterol. More particularly the inventionrelates to a rapid, efficient and stable precipitating agent forpreparing an HDL cholesterol fraction from a blood serum or plasma and aseparate reagent for measuring such cholesterol.

BACKGROUND OF INVENTION

Cholesterol levels in blood are related to the risk factors associatedwith coronary heart disease. Efforts have been made to determinecholesterol level in blood of patients but none have provided a rapidand accurate procedure.

Considerable effort has been placed on trying to identify risk factorsassociated with coronary heart disease (CHD). The clinical laboratoryhas had its share of work in this effort with respect to lipoproteinanalysis. Most laboratories offer cholesterol and triglyceride analysis,but recent studies are showing these tests to be about as obsolete astotal lipid analysis. Electrophoresis of plasma lipoproteins gave someimpetus to the presumptive pheno-typing of patients, but this testingtended to focus on abnormalities of (low density lipoproteins) (LDL or βlipoprotein) and very low density lipoproteins (VLDL or pre-βlipoproteins) with very little interest shown in the high densitylipoprotein (HDL or α lipoproteins). Clinical chemists are beginning tofocus on some of the important epidemiological studies that have shownthe probable value of HDL cholesterol estimations and show in pragmaticterms how this test can be incorporated into every routine clinicallaboratory.

One of the earliest reports associating HDL with CHD was that of Barr etal, Am.J.Med. 11,480 (1951). In this study, the researchers made theobservation that healthy men had higher HDL levels than did men withCHD. This finding was subsequently confirmed in many other studies. Morerecent publications involving large numbers of patient values have shownunequivocally the value of fractionating total cholestrol values intoHDL and VLDL and LDL cholesterol.

HDL is a lipoprotein synthesized by the liver. One proposed mechanism ofits action which fits with clinical data is that HDL is the transportmechanism which removes cholesterol from the peripheral tissues andcarries it to the liver for catabolism. Thus, if the HDL levels arenormal or high, then there is efficient removal of cholesterol whichlowers the tissue pool and lessens the risk of deposition ofcholesterol. Conversely, if the HDL levels are low, then there isinefficient removal of cholesterol and the subsequent risk of CHD ishigher. Several observations lend support to this hypothesis.

1. In Tangier disease (deficiency of HDL) all patients have excessivedeposition of cholesterol esters in the blood vessels.

2. Women have higher HDL levels than men and a lesser risk of developingCHD.

3. Blacks have higher HDL levels than whites with a lesser risk ofdeveloping CHD.

4. Children have higher HDL levels than adults, black children havehigher levels than white children.

5. The prevalence of CHD in men aged 50-69 was double in the very lowHDL groups compared to those above the population mean.

All of these studies were based on epidemiological data concerned withthe prevalence of CHD. A few studies have now been conducted asprospective studies with respect to the incidence of the disease. In areport published in 1966 researchers suggested that the incidence of CHDwas higher in young men with low HDL levels. A similar report waspublished in a study of middle aged men.

One of the most recent reports on the relationship between CHD andfasting plasma lipids was assessed by a case-controlled study in fivepopulations with a total of 6,859 men and women of black, white andJapanese ancestry. In each major study, a statistically significantinverse relationship was found with CHD: it was found in mostage-sex-race specific groups. These authors concluded that the "virtueof partitioning total cholesterol in assessing CHD risk is unequivocallydemonstrated." HDL cholesterol has a negative correlation with CHDwhereas LDL and VLDL cholesterol have a positive correlation with CHD.Hence, the "Total cholesterol (HDL & LDL & VLDL) must be a lesssensitive indicator of risk than an appropriately weighted algebraicsum." Castelli et al. Circulation, 55 767 (1977).

LABORATORY EVALUATION OF HDL CHOLESTEROL

Two basic methodologies are available for HDL cholesterol measurement.The first widely used method was preparative ultracentrifugation. Thisis a very time consuming method with low productivity yield for aroutine clinical laboratory. It also necessitates the availability ofexpensive equipment and highly trained personnel.

The second class of methods involves the precipitation of LDL and VLDLfrom serum or plasma by complexing them with a polyanion and divalentcation. Many procedures are based on precipitation of the LDL and VLDLwith heparin and Mn++. However, most of these techniques suffer from theproblems probably related to lot to lot variation in heparin. Recentliterature contains many references of attempts to improve this assay.

In an attempt to try to improve the precipitation, polyanions other thanheparin and catons other than Mn++ have been tried. One method whichuses a phosphotungstate-Mg++ complex was recently re-evaluated and wasshown to be a simple reliable method yielding results comparable toultracentrifugation analysis. Burstein et al, J. Lipid Research, 11, 583(1970).

CHOLESTEROL ANALYSIS

Measurement of serum or plasma cholesterol is technically difficultunless fairly specific analytical methods are used. It is highlydoubtful that direct serum analysis by well known Liberman Buchardanalysis for cholesterol will have the sensitivity or the accuracynecessary when dealing with low cholesterol levels found in the HDLfraction. As with most analytical methods, the closer to the limits ofthe instrumentation (such as spectrophotometers) one works, the greaterthe imprecision of the analysis. Furthermore, any interfering compoundswill show a much greater percentage of error in the nonspecific methods.These technical difficulties can be overcome by use of extractionmethods or the newer and technically simpler direct enzymaticprocedures. Indeed, the enzymatic procedures lend themselves to the typeof low level analysis since one can increase the sample size in anaqueous system.

Results of HDL cholesterol analysis should be presented to theclinicians in a usable report rather than a naked number. The reportshould describe the appearance of the serum, give total cholesterol, LDLcholesterol, HDL cholesterol, VLDL cholesterol and triglyceride values,each in mg/dl. In addition, the usual population ranges for these valuesshould be given, as follows:

    ______________________________________                                        Total Cholesterol                                                             Age            Male     Female                                                <20            <180 mg/dl                                                     20-30          140-260  140-240                                               30-40          140-280  140-240                                               40-50          140-280  150-280                                               >50            140-280  180-330                                               VLDL Cholesterol                                                              (calculated as Triglycerides/5)                                               Male and Female                                                                              0-40 Mg/dl.                                                    >40 associated with type II b, IV, or III                                     Lipoproteinemias                                                              LDL Cholesterol                                                               Male      Female                                                              62-178    66-185 mg/dl                                                        Values greater than expected range are                                        associated with a higher than average risk                                    of coronary heart disease.                                                    HDL Cholesterol                                                               Male      Female                                                              29-61     38-75                                                               Values below limits are associated with a higher                              than average risk of CHD.                                                     Values above 55 mg/dl. are associated with a                                  lower than average risk of CHD.                                               ______________________________________                                    

Total and HDL Cholesterol values are measured analytes. VLDL cholesterolis calculated by dividing the serum or plasma triglycerides by five(valid only when triglycerides are less than 400 mg/dl and when nochylomicrons are present). The LDL cholesterol is then calculated asTotal minus (HDL+VLDL) cholesterol.

SUMMARY OF THE INVENTION

Measurement of total cholesterol as an index of CHD risk has beendemonstrated to be a less sensitive approach than fractionation of thetotal cholesterol into HDL and LDL components. The LDL cholesterol has adirect relationship to CHD whereas HDL has an inverse relationship toCHD. It is now possible, using simple techniques of polyanionfractionation and simple enzymatic cholesterol determinations, toprovide clinicians with this newer tool for the assessment of risk ofCHD. These tests can provide a large data base to identify the high riskpopulation and perhaps aid in the search for factors that may elevateHDL levels thereby, hopefully favorably influence the health of thepopulation.

High density lipoprotein (HDL) cholesterol has been shown to have aninverse correlation with ischemic heart disease and may be another goodprognostic test to detect high risk subjects. Two basic methods areavailable to measure HDL. The first is ultracentrifugation analysis andthe second is selective precipitation of the LDL and VLDL lipoproteinswith divalent cations and polyanions.

The present invention is concerned with this second method of analysisbased upon the selective precipitation of (1) the low densitylipoprotein (LDL) and the very low density lipoprotein (VLDL) from (2)the high density lipoprotein to separate these two fractions foranalysis, and particularly for analysis of the HDL. As set forth in anarticle by Burstein, et al., Journal of Lipid Research, Vol. II, 1970,pp. 583-595, low density lipoproteins and very low density lipoproteins(LDL+VLDL) have been selectively precipitated from serum or plasmawithout precipitation of the high density lipoproteins (HDL) remainingin the supernatant liquid first by the addition of (1) a polyanionicpolysaccharide, or salt thereof such as heparin, dextran sulfate,mepesulfate, or sodium phosphotungstate followed by the addition of (2)a water soluble divalent cation, generally as a divalent metal salt of amonobasic acid, such as MnCl₂ or MgCl₂. It has been necessary to addeach of these required precipitants separately, in prior art methods andusing prior are reagents, since combining the two in the concentrationspreviously essential to achieve the required lipoprotein precipitationwould create an unstable precipitating reagent in which a portion of thereagent would precipitate out, generally as a divalent salt, such asMgCO₃.

The measurement of serum or plasma cholesterol is technically difficultand must be precise as set forth above. The precipitating reagents mustbe added in exactly equal quantities to the serum or plasma beinganalyzed and to assure accurate measurement of serum cholesterol againstthe standard. The necessity for two separate additions of two separateprecipitants, therefore, leaves room for error in both precipitantaddition steps.

In accordance with an important feature of the present invention, it hasbeen found that a divalent cation and a polyanion can be combined into asingle, stable precipitating reagent without reagent precipitation priorto the addition of this single precipitating reagent to thecholesterol-containing body fluid. It has been found that by combiningthe bivalent cation, in a total precipitating reagent concentration of0.25 to 0.75 molar, together with the water soluble polyanion in aconcentration of 2 to 8% based on the total weight of the liquidprecipitating reagent, the combined reagent will remain stable so thatonly one precipitation reagent addition is necessary--therebysignificantly increasing the precision of each cholesterol analysis atthe onset, in the initial separation of HDL from LDL and VLDL. Thecombined precipitating reagent including both the water soluble bivalentcation and the water soluble polyanion will remain stable and will notform a metal precipitate regardless of the particular bivalent cationand regardless of the particular polyanion so long as these twocomponents are combined in these important concentration ranges.Examples of typical water soluble bivalent cation salts are MgCl₂,MnCl₂, CaCl₂ or any other water soluble bivalent metal salt of amonobasic acid. Commonly, the MnCl₂ and MgCl₂ and CaCl₂ salts are usedin practice. Examples of typical water soluble polyanions are watersoluble salts of phosphotungstic acid, such as sodium phosphotungstate;dextran sulfate; water soluble salts of heparin, such as the sodiumsalt; mepesulfate; and chondroitin sulfate. Commonly sodiumphosphotungstate is used separately with MgCl₂ and dextran sulfate isused separately with CaCl₂ and heparin is used with MnCl₂.

After precipitation of LDL and VLDL cholesterol it is common procedureto analyze the HDL cholesterol by separately reacting the supernatantcontaining the HDL cholesterol from the serum or plasma sample with anenzymatic reagent to determine the relative color change. Cholesteroloxidase contained in the enzymatic reagent will react in the presence ofthe HDL cholesterol to produce hydrogen peroxide. Peroxidase,4-aminoantipyrine and a phenol, also contained in the enzymatic reagent,then react with the hydrogen peroxide to produce quinonemine dye (red)which is analyzed on a spectrophotometer for comparison of (1) the dyeproduced in the sample obtained from serum or plasma, with (2) the dyeproduced in the cholesterol standard sample. Typical enzymatic reagentsinclude cholesterol oxidase (microbial), cholesterol esterase (animal),peroxidase, 4-amino-antipyrine, phenol, a surfactant such as sodiumcholate, a stabilizer, and a phosphate buffer in a water solution at apH of 7.0. This enzymatic reagent is not stable for more than a fewhours, and therefore is freeze dried for later water additionimmediately prior to use. Accordingly, the enzymatic reagent must befreeze dried within a few hours after the composition is made or theentire batch is useless. Further, after the freeze dried reagent issolubilized (reconstituted) for later use in analysis, any excessenzymatic reagent which is not used within a few hours is wasted.

In accordance with an important feature of the present invention, it hasbeen found that the addition of a water soluble polyglycol having anaverage molecular weight in the range of about 190-1000 will stabilizethe enzymatic reagent for a period of seven days at a polyglycolconcentration of 0.01-0.1%, based on the total weight of enzymaticreagent, when the reagent is kept refrigerated. An enzymatic reagentwhich is stable for seven days yields tremendous advantages both inmanufacture of the enzymatic reagent and in use of the reagent toanalyze cholesterol. There is no need to hurry the initial mixing of theenzymatic reagent components prior to freeze drying and there is no needto hurry freeze drying in the manufacture of the enzymatic reagentcontaining polygylcol in accordance with the present invention. Afterreconstitution of the freeze dried enzymatic reagent, generally withfurther addition of polyglycol to supplement the glycol renderedineffective as a result of freeze drying, the reagent can be keptrefrigerated for seven days so that the analyzing laboratory need notrepeatedly and precisely reconstitute the enzymatic reagent for eachsample, resulting in imprecision and waste of expensive excess reagent.Another important benefit derived from the addition of the polyglycol tothe enzymatic reagent is that it substantially increases the activity ofthe enzymatic reagent.

Accordingly, an object of the present invention is to provide a new andimproved method of manufacturing an enzymatic reagent useful incholesterol analysis.

Another object of the present invention is to provide a new and improvedenzymatic reagent useful in cholesterol analysis.

Another object of the present invention is to provide a new and improvedenzymatic reagent useful in cholesterol analysis having dramaticallyimproved stability or useful life.

Another object of the present invention is to provide a new and improvedenzymatic reagent containing a water-soluble polyglycol surfactantcapable of rendering the reagent stable for a substantial period oftime.

These and other objects and advantages of the present invention willbecome apparent from the following detailed description of the presentinvention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

A single, stable precipitant reagent is added to whole serum or plasmato precipitate LDL and VLDL, leaving the HDL in the supernatant liquid:##EQU1##

The precipitant reagent (REAGENT 3) is a single, stable aqueous solutionof a water-soluble bivalent metal salt of a monobasic acid together witha water-soluble polyanion, generally a polysaccharide or apolyphosphotungstate. It has been found that the bivalent salt andpolyanion can be combined in a single, stable reagent so long as theconcentration of the cation is in the range of 0.25-0.75 molar and theconcentration of the polyanion is in the range of 2-8% by weight of thetotal precipitant reagent. The bivalent metal salt should besubstantially colorless in salt, i.e. magnesium chloride, with thepolyanion, i.e. phosphotungstate, in a single reagent, experimentalerror is minimized because only one addition of reagent is required andit is of larger volume which permits more accurate measurement instandard pipettes.

After addition of the precipitant reagent (REAGENT 3), the treated serumor plasma is centrifuged to segregate the precipitate (LDL and VLDL)from the supernatant liquid, containing the HDL fraction, and a precisequantity, i.e. 0.05 ml., of the supernatant is added to an enzymaticreagent capable of forming a chromophore. The enzymatic reagent containscholesterol oxidase to produce H₂ O₂, and contains 4-aminoantipyrine,peroxidase and a phenol or phenol derivative for reaction with the H₂ O₂to produce the chromophore. The reactions of the enzymatic reagent inthe presence of HDL are as follows: ##EQU2##

In addition to the essential components comprising the microbialcholesterol oxidase; animal cholesterol esterase; peroxidase; a bilesalt, such as sodium cholate; 4-aminoantipyrine; an agent, such asphenol or a phenol derivative which forms a chromophore; and a watersoluble polyglycol having a molecular weight of 190-1000, the enzymaticreagent should be at a pH in the range of 5.5 to 7.8 and thereforeincludes a buffer to maintain this pH during reaction. It may also bedesirable to add an agent useful for maintaining the solubility of freecholesterol to prevent the reagent from becoming turbid as a result ofprecipitation of free cholesterol. One suitable cholesterol solubilizingagent is a water-soluble polyglycol having an average molecular weightof about 6000 or above. The bile salt maintains a homogeneous mixture ofthe enzymatic reagent when the reagent is reconstituted with water andaids in maintaining the cholesterol in solution. The bile salt can beany alkali metal cholate, glycocholate or desoxycholate. The agentcapable of forming a chromophore can be a phenol or phenol derivativesuch as phenol, 2,4-dichlorophenol, or o-dianisidine, cresol, caryacrol,thymol, and mixtures thereof. The polyglycol is contained in theenzymatic reagent in an amount of 0.01-0.1% based on the total weight ofenzymatic reagent both before lyophilization and after the reagent isreconstituted to assure stability.

It has been found that a portion of the enzymatic reagent can beseparated and maintained as a stable liquid which can be added to a dry,powder composition containing the remainder of the enzymatic reagentwhen reconstituted for use in analysis. It has been found that thephenol or phenol derivative, bile salt, and polyglycol can be separatedand maintained stable as a separate liquid reagent (REAGENT 2) therebyreducing manufacturing costs in initially preparing a dry powdercomposition which does not require lyophilization, as an alternative toexpensive lyophilization. In this manner, an enzymatic reagent isprepared in two components: DRY FILLED REAGENT 1: microbial derivedcholesterol oxidase, animal derived cholesterol esterase, peroxidase,and 4-aminoantipyrine and a buffer suitable to maintain a pH of 5.5 to7.8, and LIQUID REAGENT 2: phenol or phenol derivative capable offorming a chromophore (yields a measurable color), bile salt such assodium cholate, and a water soluble polyglycol having an averagemolecular weight in the range of about 190-1000. Both DRY FILLED REAGENT1 and LIQUID REAGENT 2 are stable for substantial periods of time of 6months or more. To reconstitute the enzymatic reagent for use inanalysis, REAGENT 1 is dissolved in REAGENT 2 to provide an enzymaticreagent stable for seven days, when kept refrigerated (0° to 10° C.).

Alternatively, all components of the enzymatic reagent can belyophilized and later reconstituted by adding the lyophilized reagent toan aqueous solution of a water soluble polyglycol having an averagemolecular weight in the range of 190-1000.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with a preferred embodiment of the present invention, theabove-described agents are provided in kit form as four separatereagents. Reagents 1 and 2 are combined to form the working cholesterolreagent (ENZYMATIC REAGENT) to which the supernatant liquid containingHDL is added. Reagent 3 is the PRECIPITANT for the initial precipi- isprovided as the CHOLESTEROL STANDARD. Each kit contains the followingreagents for in vitro diagnostic use:

    ______________________________________                                        4 vials DRY   containing 100 U/l cholesterol                                  FILLED REAGENT 1                                                                            oxidase (microbial) (50 to 250                                                U/l.)* 50 U/l cholesterol esterase                                            (animal) (20 to 200 U/l.)* 40 ×                                         10.sup.3 U/l peroxidase (horseradish)                                         (10 × 10.sup.3 to 100 × 10.sup.3 U/l.)* 0.06%                     (w/v) 4-aminoantipyrine (.015% to                                             0.1% (w/v) solution)* phosphate                                               buffer to pH of 7.0, and a                                                    stabilizer (polyethylene glycol                                               having an average molecular weight                                            approximately 6000 or higher),                                                (1 to 2 gm./l)*.                                                4 vials LIQUID                                                                              each containing 25 ml of an aqueous                             REAGENT 2     solution containing: 0.1% (w/v)                                               phenol (0.02% to 0.2% (w/v)                                                   solution)* a surfactant, such as                                              sodium cholate (0.01% to 1.0% (w/v))*                                         and a water-soluble polyglycol                                                having an average molecular weight                                            in the range of 190 to 1000,                                                  (0.01% to 0.1% (w.v))*.                                         1 vial REAGENT 3                                                                            containing 10 ml precipitating                                                solution of 0.5 molar water-                                                  soluble bivalent metal salt of a                                              monobasic acid, such as magnesium                                             chloride (0.25 to 0.75 molar) in                                              a water-soluble polyanion, such                                               as an alkali metal phosphotungstate                                           solution (2% to 8%).                                            1 vial        containing 250 mg/dl cholesterol                                              standard in aqueous base.                                       ______________________________________                                         *Data in parentheses indicate the concentration range to achieve the full     advantage of the present invention.                                      

Reconstitution of Reagents: To prepare working cholesterol reagent(ENZYMATIC REAGENT), add the contents of one vial of REAGENT 1 to onevial of REAGENT 2. Allow approximately 10 minutes for complete solution,then swirl the vial to mix completely. The resulting ENZYMATIC REAGENTis STABLE for 7 days when refrigerated (2° to 10° C.). The workingcholesterol reagent (REAGENT 1 plus REAGENT 2) should be almostcolorless. A faint pink color is allowable if the absorbance of thesolution at 510 nm measured against water is less than 0.10. REAGENT 3and the CHOLESTEROL STANDARD are used without dilution.

Suitable polyglycols for use in initially preparing the ENZYMATICREAGENT prior to lyophilization, and for reconstitution of the ENZYMATICREAGENT include polyethylene glycols 200, 300, 400, 500, 600, 800 and1000, which are glycols of the general formula

    HOCH.sub.2 CH.sub.2 (OCH.sub.2 CH.sub.2).sub.n OH

where n is number from about 4 to about 20. The numbers 200, 300, . . .represent the approximate average molecular weight of the polyethyleneglycols. Other suitable polyglycols include aromatic ethers ofpolyethylene glycols such as Triton X-100 of Rohm and Haas Company whichis a water-soluble isooctylphenoxypolyethoxyethanol containing nine orten ethoxy groups having the general formula

    CH.sub.3 C(CH.sub.3).sub.2 CH.sub.2 C(CH.sub.3).sub.2 --C.sub.6 H.sub.4 --O(CH.sub.2 CH.sub.2 O).sub.x H

wherein the average value of x is ten. A polyglycol known as Adekatol orLeonol (a polyethoxy glycol having 16 carbon atom units) sold by LeonLaboratories is also suitable and has been found to be particularlycommercially acceptable. All of these glycols are viscous syrups whichare soluble in water at the concentrations (0.25 to 0.75 gram per liter)required for use in the present invention to provide exceptionalENZYMATIC REAGENT stability and activity.

Polyethylene glycol 200 has an average molecular weight from 190 to 210.Polyethylene glycol 300 has an average molecular weight from 285 to 315.Polyethylene glycol 400 has an average molecular weight from 380 to 420.All of these glycols are clear viscous liquids which dissolve readily inwater. Other polyglycols containing 8 to 20 carbon atoms are suitable.

The pH of the precipitant solution (REAGENT 3) is is preferablymaintained at about 7.0 although pH's of 5.5 to 7.8 are suitable.

The stabilizer in REAGENT 1 may not be essential since the polyglycol ofREAGENT 2 generally is sufficient for stabilization and cholesterolsolubilization. When a stabilizer is used in REAGENT 1, polyethyleneglycol 6000 is preferred and is present at a concentration of about 1 to1.5 g/l. Other high molecular weight polyethylene and polypropyleneglycols can be substituted for PEG 6000 so long as they arewater-soluble at concentrations of 1 g/l. The purpose of the stabilizeris to aid in solubilizing the cholesterol and is unnecessary inreconstitution because of the polyglycol used in initial manufacture oflyophilized REAGENT 1 and in reconstitution with REAGENT 2.

The surfactant in REAGENT 2 is a combination of a bile salt, such as analkali metal cholate, at a weight percentage of from 0.01% to 1.0% and apolyglycol having an average molecular weight from approximately 190 to1000 at a weight percentage of from 0.01% to 0.1%. The surfactantstabilizes the cholesterol esterase and activates the cholesterolesterase and the cholesterol oxidase.

When the combined working reagent (REAGENTS 1 plus 2), or a concentratethereof, are mixed and lyophilized (freeze-dried) at 30 microns pressureand a final temperature of 26° C., the dry product can be reconstitutedby dissolution in an aqueous solution containing 0.01 to 0.1% of awater-soluble polyglycol having an average molecular weight of 190-1000.This procedure stabilizes the enzymes throughout the resulting ENZYMATICREAGENT.

Interfering Substances: A list of potential interfering compounds hasbeen published in Young, Clin. Chem., 21, 1D-432D (1975). To date, noknown compound present in physiological concentration in serum is knownto interfere in the cholesterol analysis. Various lipoproteinemias andother dysproteinemias may interfere with the full precipitation of LDLand VLDL.

Specimen: The preferred specimen is serum following a 14-hour fast. Thepatient should have been on a full ethnic diet for several days beforeblood sampling. Serum should be stored at room temperature where it isstable for at least 5 days. Refrigeration or freezing of samples mayalter the structure of the lipoproteins yielding lower results.

Assay Procedure:

(a) Preparation of HDL Fraction

1. Pipet 1 ml serum into labelled conical centrifuge tube.

2. Add 0.1 ml REAGENT 3. Mix well.

3. Centrifuge tubes at 1000×g (full speed for most bench centrifuges)for 15 minutes.

4. Carefully remove clear supernatant fraction and transfer to alabelled tube marked HDL fraction. (Note: if the supernatant is turbidas may happen with lipemic samples, dilute serum 1/1 with saline andrepeat precipitation. Multiply the final result by 2.)

(b) Assay of Total and HDL Cholesterol

1. Label assay tubes, one for reagent blank, one for standard and onefor each sample to be analyzed.

2. Pipet 2 ml of working cholesterol reagent into each tube and place in37° C. water bath for 2 minutes to equilibrate to temperature.

3. Add the following samples to appropriate tubes: standard and wholeserum -20 microliters; HDL fraction -50 microliters.

4. Mix well and incubate tubes for 15 minutes.

5. Remove tubes from water bath and read before 30 minutes.

6. Blank photometer to zero with reagent blank at 510 nm.

7. Read and record the absorbance of each tube at 510 nm.

8. Samples greater than 500 mg/dl should be diluted 1:1 with saline andreassayed. Multiply result by 2.

(c) Calculations ##EQU3## The factor 0.447 is derived from the dilutionof the sample with REAGENT 3 and using 50 μl sample v. 20 μl standard.##EQU4##

Expected Values: One study performed using this kit resulted in a rangeof 30-75 mg/dl for HDL cholesterol and 130-280 mg/dl for totalcholesterol.

Performance Characteristics: Precipitation of LDL and VLDL lipoproteinsassessed by electrophoresis was 100%.

Linearity of the cholesterol assay was to 500 mg/dl.

The absorbancy of the 250 mg/dl cholesterol standard was 0.390±0.03.This will vary with the accuracy of the micropipet used.

Precision studies of a lyophilized control serum analyzed daily for 20days was: mean 230 mg/dl and C. V. 2.2%.

EXAMPLE 1

Reagent 1 is a dry powder containing 100 units per liter of cholesteroloxidase, 50 units per liter of cholesterol esterase, 40,000 units perliter of peroxidase, 0.06% (w/v) 4-aminoantipyrine and 1.15 grams perliter of polyethylene glycol 6000 as stabilizer. Phosphate buffer (14.2g. Na₂ HPO₄ and 7.5 g. KH₂ PO₄ per liter) is included to adjust the pHof the reagent to a value between 6 and 8, preferably 7.

Reagent 2 is produced by dissolving 2 grams of sodium cholate in 900 mlof distilled water. Then 0.5 ml of polyethylene glycol or molecularweight from 190 to 1000 is dissolved in the solution, followed by 1.1 mlof a 90% aqueous solution of phenol.

Reagent 3 is made by dissolving 40 grams of phosphotungstic acid in 700ml of distilled water. To the resulting solution is added 60 ml of a 10%(w/v) solution of NaOH and 100 grams of MgCl₂.6H₂ O. The solution isthen diluted to one liter volume. Its pH is 6.8±0.2.

Reagents 1 and 2 are admixed to form the cholesterol reagent, which isstable for at least a week when refrigerated (2° to 10° C.).

What is claimed and desired to be secured by Letters Patent of theUnited States is:
 1. A stable, aqueous enzymatic reagent for interactionin the presence of cholesterol to provide a measurable chromophore, saidreagent capable of remaining stable for a relatively long period of timecomprising:effective amounts of cholesterol oxidase, cholesterolesterase derived from an animal source, peroxidase, 4-aminoantipyrine,an agent capable of forming a chromophore, a bile salt, a water-solublepolyglycol having an approximate weight average molecular weight in therange of 190-1000 selected from the group consisting of polyethyleneglycol and polyethoxy glycol, in an amount of 0.1-1.0 grams per liter ofenzymatic reagent and a stabilizer comprising a water-soluble polyglycolhaving an approximate weight average molecular weight of 6000 or higherin an amount sufficient to maintain solubility of free cholesterol, saidenzymatic reagent having a pH in the range of 5.5 to 7.8.
 2. Anenzymatic regent as defined in claim 1 wherein said polyglycol having anapproximate weight average molecular weight of 6000 or higher ispolyethylene glycol.
 3. An enzymatic reagent as defined in claim 1wherein said agent capable of forming a chromophore comprises a phenolor a phenol derivative.
 4. An enzymatic reagent as defined in claim 1wherein said chromophore forming agent comprises a phenol or phenolderivative.
 5. A two component enzymatic reagent comprising a first,solid, enzymatic composition and a second, separated, liquid activatortherefor, both the solid enzymatic composition and the liquid activatorbeing substantially stable for a period of time of 6 months or more whenseparated, comprising:a first, solid enzymatic composition comprisingenzyme effective amounts of cholesterol oxidase, cholesterol esterasederived from an animal source, peroxidase, 4-aminoantipyrine and awater-soluble polyglycol having an approximate weight average molecularweight of 6000 or higher in an amount sufficient to maintain solubilityof free cholesterol, said composition having a pH in the range of 5.5 to7.8; and a second, liquid activator comprising a bile salt and a watersoluble polyglycol having a weight average molecular weight in theapproximate range of 190 to 1000 selected from the group consisting ofpolyethylene glycol and polyethoxy glycol.
 6. An enzymatic regent asdefined in claim 5 wherein said polyglycol having an approximate weightaverage molecular weight of 6000 or higher is polyethylene glycol.
 7. Amethod of manufacturing an enzymatic reagent having increased stabilitywhen reconstituted with water comprising admixing an aqueous solution ofcholesterol oxidase, choletserol esterase derived from an animal source,peroxidase, 4-aminoantipyrine, a bile salt, a water-soluble polyglycolhaving an approximate weight average molecular weight of 6000 or higherin an amount sufficient to maintain solubility of free cholesterol, anda lower molecular weight water-soluble polyglycol having a weightaverage molecular weight in the range of about 190 to 1000 selected fromthe group consisting of polyethylene glycol and polyethoxy glycol, saidenzymatic reagent having a pH in the range of 5.5 to 7.8, said lowermolecular weight polyglycol being present in said solution in an amountof 0.01%-0.1% by total weight of said solution; anddrying said aqueoussolution to provide a substantially completely solid enzymaticcomposition.
 8. A method as defined in claim 7, wherein said polyglycolhaving an approximate weight average molecular weight of 6000 or higheris polyethylene glycol.